The long-term objective of this proposal is to understand the molecular events that lead to the determination and differentiation of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered numerous transcription factors and signaling molecules as pivotal regulators of retinogenesis. These factors are found to act at various developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, both transcription factors and signaling molecules play essential roles in controlling cell specification and differentiation during retinogenesis. Despite these important advances, however, the molecular targets and signaling events downstream from many transcription factors still remain poorly understood and the interplay between these two classes of molecules remains largely to be determined. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by Dll4-Notch signaling as well as on the genetic relationship between Dll4 and the Foxn4 winged- helix transcription factor. Our preliminary data and previous work have led us to propose that Dll4 may have a specific role in mediating Notch signaling in the specification and differentiation of retinal cell types and that it may act as a direct target of Foxn4 to mediate part of its function during retinogenesis. This application primarily aims to test this hypothesis by pursuing four specific aims: i) to test Foxn4 as a direct upstream regulator of Dll4 expression. Our goal is to confirm the specificity of Foxn4 in regulating Notch signaling molecules and to investigate whether Foxn4 has the ability to directly bind and activate the Dll4 promoter by a combination of molecular, bioinformatic and biochemical approaches;ii) to study the biological function of Dll4 during retinogenesis by conditional gene targeting. We aim to generate retina- and stage-specific Dll4 knockout mice and functionally characterize them at anatomical, cellular and molecular levels to identify the developmental and cellular processes that Dll4-Notch signaling regulates;iii) to investigate Dll4-Notch signaling events during retinogenesis by overexpression analysis. We aim to employ an overexpression approach to determine the presumed cell-autonomous and non-cell-autonomous effects of Dll4 and its dominant-negative form on retinal cell specification and differentiation;and iv) to map the fate of Dll4- expressing retinal progenitors. Our goal is to determine the lineages of Dll4-expressing retinal progenitors using the Cre-loxP fate-mapping strategy. The proposed studies together are expected to provide important insights into the regulatory gene network and signaling events that underlie the determination and differentiation of different retinal cell types. PUBLIC HEALTH RELEVANCE: The proposed studies aim to elucidate the genetic regulatory network and signaling events involved in retinal cell specification and differentiation, and thus will lay the foundation for a better understanding and treatment of blinding neuroretinal diseases. In particular, they are highly relevant to achieving controlled regeneration of desired retinal cell types from stem cells for therapeutic purposes.